With a high attributable mortality rate of 40-50%, Candida species represent the fourth leading cause of hospital-acquired bloodstream infections in the United States. These fungal pathogens are normally found as commensals in mammalian mucosal niches, especially the oral cavity. In immunocompromised patients, Candida overgrowth leads to the formation of biofilms, which play an important role in the development of infection. Biofilms are complex microbial communities that adhere to a surface and are enclosed within an extracellular matrix. Many studies have shown that biofilms are generally more resistant to antimicrobial agents than single, planktonic cells. These biofilms are associated with the opportunistic yeast infection, oral candidiasis (oral thrush), found in newborns and many patients with AIDS, diabetes and cancer. Even though C. albicans is the predominant organism found in oral thrush, there is an increasing incidence of oral thrush isolates consisting of non-albicans Candida species (especially C. glabrata, C. dubliniensis and C. tropicalis). This finding is clinically relevant since conventional doses of antifungals are sometimes ineffective at clearing these oral infections. While single-species Candida biofilms have been well-studied, considerably less is known about the composition and interaction of Candida species in mixed biofilms in response to antifungal treatment. I have recently developed a highly accurate quantitative-PCR-based approach to determine the precise species composition of mixed Candida biofilms. Preliminary data shows that the proportion of C. glabrata in the mixed biofilm increases significantly upon treatment with fluconazole. My preliminary data also indicates that the metabolic activity of biofilms is altered when these four Candida species are mixed under different nutritional environments, suggesting that biofilm composition and dynamics have changed as well. Based on this data, my hypothesis is that the shift in mixed Candida species biofilm composition and spatial architecture following antifungal treatment favors non-albicans Candida species that are intrinsically resistant or rapidly develop drug resistance. In order to test this hypothesis, I will perform experiments to address the following specific aims: (1) To determine the precise composition of mixed Candida species biofilms generated from susceptible and drug-resistant strains in the presence vs. absence of antifungal treatment and (2) To determine the spatial architecture of Candida species in mixed biofilms generated in the presence and absence of antifungal treatment. Collectively, these innovative studies will have significant impact since they will provide new information about how mixed Candida species biofilms are associated with antifungal resistance. These studies will also test the efficacy of my quantitative-PCR assay to serve as a diagnostic tool for rapid composition determination of mixed Candida biofilms (i.e. oral thrush). Since mixed Candida species biofilms are encountered more frequently, these studies will pave the way for the development of more effective antifungals to improve patient outcomes. Moreover, successful completion of this project will provide exceptional training, advance my career development and prepare me to become a pediatric dental clinician scientist.

Public Health Relevance

Candida albicans is most common cause of oral fungal infections worldwide. However, there is an increasing incidence of oral infections consisting of other non-albicans Candida species that rapidly develop resistance to common antifungals. These studies will provide new and important information about how the composition and physical arrangement of different Candida species in mixed biofilms is associated with antifungal resistance, leading to the development of more effective drug therapies to improve oral health and patient outcomes.